PNK catalyzes the transfer of the γ-phosphate of ATP to the 5′-hydroxyl group of DNA, RNA, shorter oligonucleotides and nucleotide-3′-phosphates. Because of this activity it is widely used in molecular biological methods. The activity of PNK is measured for the purpose of assessing the quality of enzyme preparations. Typical methods utilize γ-32P ATP as the phosphate source and determine the amount of the radiolabel incorporated into the product by such means of thin layer chromatography followed by scintillation counting. Quantitative assays involving phosphorylation of polynucleotides or oligonucleotides followed by ligation and detection of ligated products have not been reported to the best of Applicant's knowledge.
Numerous enzyme-linked assay methods have been devised for the sensitive detection of a wide variety of analytes. Enzyme-linked methods are now widely used in the fields of immunoassay and DNA probe assays. The chief impetus behind the development of these methods is the improvement of sensitivity due to signal amplification afforded by the catalytic turnover of substrate to produce a detectable product. The most commonly used enzymes are alkaline phosphatase, β-galactosidase and horseradish peroxidase (L. J. Kricka, Ligand-Binder Assays, Marcel Dekker, Inc., New York, 1985, Chapter 6, pp. 165–198; C. A. Dangler, Nucleic Acid Analysis, Wiley-Liss., New York, 1996, Chapter 3, pp. 47–66). PNK does not appear to have been used as a label in these types of assays.
The enzymatic ligation of pairs of oligonucleotides bound to a target nucleic acid is a widely used method, finding application in for example the ligase chain reaction (LCR), methods for circularizing nucleic acids and in hybridization assays where a capture oligonucleotide is linked to a signal oligonucleotide when hybridized to a target. It is generally thought that the oligonucleotides must each be of a minimum length to be ligated efficiently. Recent work has shown this minimum length to be about 6–8 bases (C. E. Pritchard and E. M. Southern, Nucl. Acids Res., 25, 3403–3407 (1997)). Typically, much longer nucleic acids are employed.
Applicant's U.S. Pat. Nos. 5,998,175, 6,001,614, 6,013,346 and 6,020,138, which are fully incorporated herein by reference, disclose methods for sequentially ligating a plurality of oligonucleotides onto a hybridized “primer” or anchor oligonucleotide in one step under conditions where only the anchoring oligonucleotide is stably hybridized. An advantage of this sequential multiple ligation technique is that many detectably labeled units are able to be added onto the product nucleic acid under highly controlled conditions. Ligation only occurs when the anchoring oligonucleotide is hybridized and all oligonucleotides are of the correct sequence to align in a contiguous manner on the template.